Bacteriophages are highly specific viruses that infect bacteria. Following infection of a bacterium like E. Coli by a lytic phage, such as T4, a profound rearrangement of all macromolecular syntheses occurs. The RNA Polymerase (RNAP) of the host bacterium binds to the initiation sites of the phage genome known as Immediate-Early (E) genes and transcribes them. Some of the IE gene products degrade the host (bacterial) DNA which lacks the modified base Hydroxy Methyl Cytosine (HMC) while another product ADP-Ribose, binds to the alpha subunits of the bacterial RNAP and renders it incapable of recognizing bacterial cell promoters. This results in the cessation of transcription of host genes. These events occur in the first 3 to 5 minutes after infection.
In the next stage, the modified RNAP recognizes and binds to the so-called Delayed Early (DE) genes, thus eliminating further expression of the IE genes of the phage. The DE gene products are involved in replicating the phage genome using the degraded bacterial DNA bases. One of the products of the DE genes is a novel sigma factor that causes the host RNAP to recognize only the Late (L) genes which are the next to be transcribed. The Late genes are involved in synthesizing new capsid proteins, tails and tail fibers and assembly proteins all of which are needed to assemble progeny phage particles. Finally, the phage lysozyme gene is activated resulting in the lysis of the bacterial host cell and release of the progeny phage.
In view of their highly specific lytic effect, bacteriophages acting on infectious pathogens have been investigated from the time of their discovery to the present day for their therapeutic potential. Soon after their discovery in 1915–17 (d'Herelle. Crit. Rev. Acad. Sci. Paris, 165, 373 (1917)), bacteriophages were used extensively in both the U.S. and Europe for the treatment of bacterial infection. Bacteriophage preparations for treatment of bacterial infections (see, e.g., U.S. Pat. No. 6,121,036) and in inhibition of dental caries (U.S. Pat. No. 4,957,686) have been described. Although highly successful initially, phage therapy are controversial due to lack of quality control, regulatory processes and inadequate understanding of the high specificity of phages for their bacterial hosts. Phage therapy was abandoned in the western world after the advent of antibiotics in the forties. However, in view of the emergence of antibiotic resistance in recent years, there is renewed interest in the development of phage therapy for treating infection (Sulakvelidze et al. Antimicrob Agents Chemotherap, 45, 649, (2001)).
Although the efficacy of phage therapy is widely recognized, there are several problems that need to be addressed before phages can become acceptable therapeutic agents. Many of the problems encountered by the early investigators, such as removal of host bacteria and bacterial debris from therapeutic phage preparations, can be overcome by modern methodologies that have been developed in the past few decades. Basic properties of phages like rapid clearance by the spleen, liver and the reticulo-endothelial system, and the potential for development of antibodies in the human host during treatment, however, require novel solutions if phage therapy is to become generally applicable. One approach for addressing the first problem, namely, rapid clearance, was described by Merrill et al (Proc. Natl. Acad. Sci. USA 93, 3188 (1996) see also U.S. Pat. No. 5,688,501) which involved the selection of long-circulating variants of wild type phages by serial passage in animals.
The generation of neutralizing antibodies after the administration of phages to humans and animals is one of the major concerns that hinders the development of phage therapy, especially for chronic infections. It has been reported that neutralizing antibodies appear a few weeks after the administration of phages to humans or animals (Slopek et al. Arch. Immunol. Ther. Exp., 35, 553 (1987). Administering higher doses of phage has been suggested as a possible solution (Carlton, R. M., Arch. Immunol. Ther. Exp., 47, 267 (1999); however, this is not the most attractive of alternatives. For example, a high-dosing approach requires production of a far greater number of phage for each dose to be administered.
Many studies of potentially therapeutic phages to date have focused on the lytic endpoint that releases progeny phage which can invade other bacterial hosts and destroy them. This amplification provided by the lysis of the bacterial host is an attractive feature of phage therapy, as it facilitates production of more phage and killing of infecting bacteria. However, phage amplification and release through lysis also exposes the subject being treated to a bolus of bacteriophage. This poses the risk that the host will mount an immune response to the phage, which immune response may be undesirable, facilitate clearance of the phage, or both.
During the past decade, the key components essential for host lysis by bacteriophages have been investigated. It is now recognized that two proteins, an endolysin and a holin are needed for host lysis to occur. Endolysins are muralytic enzymes that accumulate in the cytosol and holins are small membrane proteins that regulate access of the endolysins to the cell wall through the cytoplasmic membrane (Wang et al. Ann. Rev. Microbiol. 54, 799–825 (2000)). The lysis gene region of bacteriophage lambda was cloned into a multi-copy plasmid, pBH 20 under the transcriptional control of the lac operator and induction of this “lysis operon” led to lytic behavior parallel to that of bacteriophage infected cells (Garrett, J. et al. Mol. Gen. Genet. 182, 326 (1981). The two lysis genes cph1 and cpl1 of the Streptococcal pneumoniae bacteriophage Cp-1, coding for holin and lysin respectively, have been cloned and expressed in E. coli (Martin et al. J. Bacteriol. 180, 210 (1998)). Expression of the Cph1 holin resulted in bacterial cell death but not lysis. Concomitant expression of both holin and lysin of phage Cp-1 in E. coli resulted in cell lysis. Furthermore, the cph1 gene was able to complement a lambda Sam mutation (carrying an amber mutation in the holin gene) in the nonsuppressing E. coli HB101 strain to release phage progeny. Regulated expression of lambda phage lysis genes S and R causes dramatic lysis of both Vibrio cholerae and Salmonella enterica serovar Typhimurium cells (Jain et al. Infect Immun, 68, 986 (2000).
There is a need in the field for methods and compositions to provide for therapeutic bacteriophage having reduced immunogenicity, and thus reduced clearance, in the host. The present invention addresses this need.